Poliovirus extraction

Cannahinoids are usually well tolerated, and do not produce the generalized toxic effects of conventional chemotherapies . Anti-ulcer activity. Petroleum ether extract of the dried aerial parts, administered intra-peritoneally to male rats, was active k Antiviral activity. Hot water extract of the dried fruit, in veto cell culture at a concentration of 0.5 mg/mL, was inactive on herpes simplex 1 virus, measles virus, and poliovirus... 

Antiviral activity. Hot water extract of the seed, in cell culture, produced weak activity on poliovirus 1 ... 

Antiviral activity. Water extract of the dried fruit, in cell culture at a concentration of 10%, was inactive on herpes virus-type 2, influenza, poliovirus, and vaccina . Tincture of the dried fruit, administered orally to adults of both sexes at a dose of 60 mL/person, was active on herpes virus. PCT patent number W096/14064 reported treatment of six subjects with herpes virus infections. The activity was highly dose... 

Fig. I. Examples of Western blots stained with colloidal gold for total protein followed by immunostaining of individual antigensJ.anes 1—3 Proteins on a Western blot from a cytoplasmic extract of poliovirus-infected HEp-2 cells were stained with colloidal gold. The probing monoclonal antibodies, recognizing the viral proteins VP1 and precursor, VPO and VP2, and VP3, respectively, are detected by peroxidase-coupled rabbit-antimouse antibody (asterisks). Lane 4 Western blot of an E. coli lysate, containing a fusion protein composed of protein A and the poliovirus protein 2B. The fusion protein (arrowhead) is detected on the gold-stained blot by peroxidase-coupled IgG that binds to the protein A moiety.

The hot water extracts of bulbs and leaves of Haemanthus albiflos had strong antiviral activity against Poliovirus 1, Herpes simplex 1 virus. Vesicular stomatitis vims and simian Rotavirus SA11 (100, 101). The bulbs of this species also showed strong antiviral activity against Moloney murine leukemia virus and HIV(i02). 

If purified poliovirus is not substantially concentrated at a trichloro-trifluoroethane (C2Cl3F3)-water interface, since fluorocarbons are among the most hydrophobic substances known, it is rather improbable that hydrophobic interactions could be involved in poliovirus adsorption to any material. This is particularly true with our oxide surfaces, which in being wet with water, are demonstrated to be hydrophilic in character. This C2CI3F3 extraction procedure is commonly used in enterovirus purification, and is highly effective in removing hydrophobic materials (for example, lipids) from partially purified preparations. 

Dilute, highly purified poliovirus, which would occupy less than 0.03 cm if hexagonally closest packed in a two dimensional array, was emulsified with equal volumes (10 mL) of 0.02 Z, pH 7 buffer and C2CI3F3 in borosilicate extraction tubes at 25°C. The emulsion was allowed to separate spontaneously, which took about 5 min, and the aqueous phase was sampled for residual virus. In Table IX we see only a small decrease in infectivity and radioactivity in the C2Cl3F3-extracted samples, compared to controls run in parallel but not containing C2CI3F3. This experiment was repeated and essentially identical results were obtained. 

The presence of informosomelike RNPs has been demonstrated also in the case of Ehrlich ascites carcinoma cells infected with Sendai virus (Volkova et al., 1969). The cytoplasmic extracts of the cells labeled for 30 minutes with uridine contain virus-specific RNA in the form of particles with a sedimentation coefficient 45S (the sedimentation coefficient of the complete virus equals 57S). The buoyant density of the RNP peak in CsCI equals 1.43 to 1.44 g/cm. Although these properties are compatible with the idea that they are informosome, the particles, and in particular their protein component, should be characterized in more detail before reaching a definite conclusion. Recently SOS virus RNA-containing particles with p = 1.40 g/cm have been found in HeLa cells infected with poliovirus (Huang and Baltimore, 1970), although the authors have some doubts about the reality of these complexes. 

Most recently Rose et al. (55) have identified an initiation factor that is inactivated after infection of HeLa cells with poliovirus. In their experiments Rose al. (55) took advantage of the finding that translation of YSV mRNA, like host mRNA translation, is inhibited in cells superinfected with poliovirus (52, 56). They prepared extracts from poliovirus-infected and uninfected HeLa cells, and after a preincubation period and RNase treatment to eliminate endogenous mRNA translation, tested the ability of the extracts to translate exogenous poliovirus and 7SV mRNA. Poliovirus mRNA was translated by both extracts, but YSV mRNA was translated only in the extracts from uninfected cells. 

Their results further indicated that extracts from infected cells contain an activity that causes the slow inactivation of eIF-4B. Apparently, the translation of poliovirus mRNA in cell-free systems requires little or no eIF-4B, or can use an altered form of... 

In summaxy, the available evidence suggests that an initiation factor is inactivated after infection of HeLa cells by poliovirus, possibly by a virus-coded factor. The variety of results seen in comparative studies using extracts from infected and uninfected cells in terms of translation of cellular and viral messages is not understood, but may be a property of the virus or cell system under study. 

In this review I have outlined several theories that have been proposed to explain the mechanism by which picornaviruses inhibit cellular protein synthesis. Some theories seem less likely than others. Inhibition by ds ENA, for example, is no longer thought to be a likely possibility. In cell-free extracts ds ENA inhibits both cellular and viral mRNA translation (61). The inhibitor of cellular protein synthesis would be expected to be selective in its inhibitory activity. It is also apparent that picornavirus infection does not result in the degradation or alteration of cellular mENA (9> 27, 29 51). So,.too, experiments demonstrating that protein synthesis inhibition takes place in the absence of significant viral ENA synthesis (I4) tend to weaken the argument that protein synthesis inhibition results from direct competition of viral mENA with cellular ENA for initiation factor eIE-4D (47) As mentioned earlier, superinfection with poliovirus of cells infected with VSV prevents VSY mENA translation (J2, 56). In lysates from uninfected HeLa cells, however, 7SY mENA translation is favored over poliovirus mENA translation when both mENA species are present in equimolar saturating concentrations (55) If competition were a major cause of cellular protein synthesis inhibition, one would have expected poliovirus mENA to out-compete VSV mENA in cell-free translation, not the contrary. 

HELENTJARIS, T. and EHRENFELD, E. Control of protein synthesis in extracts from poliovirus-infected cells. I. mRNA discrimination by crude initiation factors. J. Virol. (1978),... 

Under properly defined conditions picomaviruses interrupt host RNA and protein synthesis (1 ) and subvert the cellular machinery to production of viral protein and ENA. By feeding radiolabeled amino acids to virus-infected cells after cessation of host-protein synthesis, viral protein can be selectively labeled. In a pioneering study, which introduced the now widely used SDS-polyacrylamide gel electrophoresis technique. Summers et al. (2) identified some 14 different virus-specified polypeptides in extracts of poliovirus infected HeLa cells. The net mass of these polypeptides exceeded two-fold or more the known coding capacity of the viral genome. 

Approximately 25% of the total polysomes in poliovirus-infected and uninfected HeLa cells are membrane-bound (10). The membrane-bound polysomes are about five times more active in translation per unit mass than free polysomes, as determined by incorporation of amino acids in cell-free extracts (11). The exact role of the membrane in this process has not yet been defined. 

Recent evidence points to the presence of protease activity-associated with polysomes and ribosomes when extracts of uninfected cells are assayed (refs. 27 32, Figure j). Characteristic of infection of cells by poliovirus is drastic, rapid inhibition of protein synthesis. Poliovirus infection also depresses the ribosomal protease activity (27, 29, 55) Ribosomes from uninfected cells have been reported to possess an autoproteolytic activity (31, 32), and this has been confiimed by two-dimensional gel analysis (Figure 4) Poliovirus infection of HeLa cells reduces the autoproteolysis of isolated 808 ribosomes markedly (not shown). The inhibition of HeLa cell ribosomal protease activity requires protein synthesis, but proceeds in the presence of guanidine (55) ... 

Figure 7 Stability of poliovirus protease in heated cell extracts. Protease extracted from amino acid analog treated cells or from control cultures was heated at 55 for up to one hour, then chilled to 0, and assayed at 55° Untreated...

A poliovirus mutant was previously described(l, 29, 33), which displayed a lack of regulation of viral RRA metabolism. Extracts of cells infected with this virus display lower amounts of protease than do those of wild type virus. Moreover, the protease produced by the mutant virus is more temperature-sensitive than that of its parent (Figure 12). These resiilts indicate that the protease itself is coded for by the viral genome. 

Figure 11. Protease activities detected in extracts of HeLa cells infected with either poliovirus type 1 (Mahoney) or defective-interfering mutants, the latter supplied by A. Nomoto and E. Wimmer, SUNY, Stony Brook, New York.

Inhibitors of well known metallo-, sulfhydi yl and serine proteases block viral proteolysis in infected cells. The use of iodoacetamide (lAM) to stabilize poliovirus and foot-and-mouth virus precursors has been described (36, 52). The preciirsors were subsequently cleaved in vitro to yield normal-sized products (36)1 and polio RHA replicase activity was stabilized in cell-free extracts of lAM-treated cells (55) H-ethyl maleimide inactivates an EMC virus-specific protease in reticulocyte lysates (20). These results could signify that a sulfhydryl-type protease, e.g., cathepsin B, is required for some of the cleavage reactions. However, when such compounds are added to cells, numerous proteins react, and there is substantial inhibition of protein synthesis. Energy metabolism in such cells is depressed, and ATP-reqiiiring proteolytic reactions blocked. 

Other investigators have studied translation of poliovirus ENA in non-preincubated cell-free systems prepared from infected HeLA cells, in which ribosomes initiate and elongate on endogenous viral mRNAs which were in the cell at the time of extract preparation (32, 33) These extracts synthesize polypeptides which show excellent correlation with those made in infected cells (32, 34, Ehrenfeld, unpublished observations). Neither detectable premature termination nor cleavage deficiencies are apparent. 

Analysis of a second picornavirus was deemed important in light of the conflicting data from two laboratories, to demonstrate that the utilization of two initiation sites for vitro translation was not unique to a particular laboratory virus stock. Consequently, experiments similar to those described above were performed with protein-synthesizing extracts from LSc poliovirus-infected cells. The LSc strain is a multi-step, temperature-sensitive mutant derived from the Mahoney strain of poliovirus type 1. It was selected for these studies because previous work had suggested that initiation of translation of LSc virus proteins vivo showed a greater resistance to hypertonic salt treatment than did translation of Mahoney virus proteins (39)i and other workers have... 

The biological significance of the existence of two initiation sites for translation of poliovirus RNA In vitro depends upon whether or not these sites are utilized for translation vivo. Although all of the identified translational products do appear to be made in equal molar amounts, when corrected for alternate cleavage pathways and protein degradation (45 "47> said see chapter 7 of this book), the relative synthesis of Wg (which has not been detected free of RNA in the cytoplasmic extracts of infected cells), has not... 

Actiially, the first demonstration that the infectivity of an animal virus resided exclusively in its ENA was provided by Colter al in 1957 (l6)s Experimental evidence was then produced that the MA extracted from mengovirus-infected Ehrlich ascites cells was infectious. This observation was soon extended to poliovirus RNA (17). Retrospectively considered, it was a very happy circumstance indeed that this pioneer work was carried out in the picornavirus system, for we now know that similar experiments performed with other RRA-containing animal viruses (e.g. influenza) would never have produced such a clearcut result. 

The total synthesis of a sequence of poliovirus 1 (Mahoney) [PVl (M)] is divided into three stages synthesis and assembly of fiill-length poliovirus-specific cDNA, transcription of cDNA into RNA, and generation of virions in a cell-free extract (Fig. 2). 

Translation mix (7.62x) 1 mM ATP, 63 pM GTP, 26 pg/ml creatine phosphate (Sigma, 10 mg/ml in 10 mM K-HEPES, pH 7.4), 20 mM K-HEPES, 20 mM calf liver tRNA, 13 pM amino acid mix, 263 pM spermidine (in 10 mM K-HEPES, Sigma), and 250 pi distilled water. For analyzing the products of in vitro translation of poliovirus RNA in HeLa cell-free extract, the amino acid mix used for the translation mix is not supplemented with methionine. For production of poliovirus in HeLa cell-free extract, the amino acid mix is supplemented with methionine. Aliquot mix and store at -80°C. Individual components should be stored at -20°C. 

To analyze the products of in vitro translation of polio RNA in HeLa cell-free extract, the master mix for PVM translation (sufficient for ten reactions) is prepared as follows 50 pi HeLa cell-free extract, 17 pi translation mix (minus methionine), 9.17pll0x salt mix, 8.3 pi S-methionine, 1 pi RNase inhibitor, and 6.15 pi distilled water. Master mix for the synthesis of poliovirus in HeLa cell-free extract is prepared as follows 50 pi HeLa cell-free extract, 17 pi translation mix (supplemented with methionine), 9.17 pi lOx salt mix, 1 pi RNase inhibitor, and 14.45 pi distilled water. 

---